Arachidonylethanolamide (AEA) has recently been isolated from porcine brain, binds with high affinity to the cloned cannabinoid receptor, mimics pharmacologic effects of cannabinoids, and has been proposed to be the endogenous cannbinoid receptor ligand. Quantitative measurements of AEA could provide important evidence for this proposed role, for example by demonstrating codistribution of AEA and of cannabinoid receptors in various brain regions. We have prepared [2H8]-labeled AEA and fluorinated AEA derivatives for use in isotope dilution negative ion mass spectrometric measurements. These methods can detect subpicomole amounts of AEA, have been used to demonstrate AEA production by rat tissue homogenates in vitro from exogenous arachidonic acid and ethanolamine, and should be capable of measuring AEA levels in rat brain if those levels were comparable to those isolated from porcine brain (ca. 380 pmol/g). No detectable levels of AEA (< &5 pmol/g) were observed in fresh rat cerebrum or cerebellum whether or not the tissues were processed in the presence of inhibitors of AEA hydrolysis. AEA (> 1 nmol/gram of tissue) was produced during alkaline hydrolysis of tissue phospholipid extracts. This appears not to reflect hydrolysis of an N-arachidonyl-ethanolamine phospholipid (NAPE) precursor of AEA because Streptomyces chromfucsis phospholipase D, which is active against NAPE, failed to generate AEA from tissue phospholipids despite substantially complete conversion of tissue phospholipids to phosphatidic acid. Such experiments suggested that the abundance of NAPE in fresh rat brain is less than 1 part per million phospholipid molecules. That the AEA generated during alkaline hydrolysis of tissue phospholipids arose from base-catalyzed aminolyisis of arachdionate-containing glycerolipids was suggested by the